Erythropoietin regulates developmental myelination in the brain stimulating postnatal oligodendrocyte maturation

Myelination is a process tightly regulated by a variety of neurotrophic factors. Here, we show—by analyzing two transgenic mouse lines, one overexpressing EPO selectively in the brain Tg21(PDGFB-rhEPO) and another with targeted removal of EPO receptors (EPORs) from oligodendrocyte progenitor cells (OPC)s (Sox10-cre;EpoRfx/fx mice)—a key function for EPO in regulating developmental brain myelination. Overexpression of EPO resulted in faster postnatal brain growth and myelination, an increased number of myelinating oligodendrocytes, faster axonal myelin ensheathment, and improved motor coordination. Conversely, targeted ablation of EPORs from OPCs reduced the number of mature oligodendrocytes and impaired motor coordination during the second postnatal week. Furthermore, we found that EPORs are transiently expressed in the subventricular zone (SVZ) during the second postnatal week and EPO increases the postnatal expression of essential oligodendrocyte pro-differentiation and pro-maturation (Nkx6.2 and Myrf) transcripts, and the Nfatc2/calcineurin pathway. In contrast, ablation of EPORs from OPCs inactivated the Erk1/2 pathway and reduced the postnatal expression of the transcripts. Our results reveal developmental time windows in which EPO therapies could be highly effective for stimulating oligodendrocyte maturation and myelination.

overexpressing human EPO only in the brain under the platelet-derived growth factor B promoter (Tg21, PDGFB-EPO) 26,27 , we have previously shown that EPO accelerates the neuronal maturation of GABAergic and glutamatergic neurons in the hippocampus 28 .We also showed that EPO activates the extracellular signal-regulated kinase 1/2 (Erk1/2) and AKT pathways in neurons, stimulates brain mitochondrial function, increases mitochondrial mass and vesicle numbers in the synaptic terminals and enhances cognition 29 .EPO-mediated Erk1/2 activation, GABAergic signaling, and mitochondrial function allegedly influence the initiation of myelination [30][31][32] .However, it is still unknown whether, during postnatal brain development, EPO is also a signaling pathway for stimulating oligodendrocyte maturation and myelination.Here, we evaluated in mice, the postnatal expression of EPOR in the subventricular zone (SVZ) of the lateral ventricle, an area of oligodendrogenesis, and found a very high postnatal expression.Thus, we aimed to investigate the role of EPO in developmental myelination and its regulation in oligodendrocyte progenitor cell (OPC) proliferation and differentiation.We analyzed two transgenic mutant mice, the Tg21,PDGFB-EPO overexpressing EPO preferentially in neuronal cells without alterations in systemic EPO expression 26,27 , and one with targeted ablation of EPORs from OPCs (Sox10-cre;EpoR fx/fx ) 33 .
To further investigate the impact of EPO on myelination, we performed an ultrastructural analysis in the white matter at P11 and P14 (n = 6 mice/group), focusing on the initial formation of the myelin sheath around callosal axons.At P11, 150 myelinated axons in WT and 111 in Tg21 mice were analyzed.At P14, 1464 myelinated axons in WT and 842 in Tg21 were analyzed.Although the total number of myelinated axons could not be determined for this area because numerous profiles could not be identified unambiguously, an almost ten-fold increase in the number of myelinated axons was quantified at P14 in a similar number of sections.The frequency distribution analysis of myelinated axonal diameters showed no correlation between axonal diameter and myelin sheaths nor difference across genotypes at either age (Fig. 3B,C, left panels, 2-sample KS: 0.1998 and KS: 0.218).A genotype difference was seen in the number of myelin layers at P11 and P14, with a larger fraction of axons having more myelin layers in the Tg21 mice (Fig. 3B,C; middle panels, KS-Test, P = 0.04, and P < 0.001), suggesting that the formation of the myelin sheaths occurs faster in Tg21 mice than in WT mice.The enhanced number of layers in Tg21 is also reflected by thicker myelin layers, as shown by the reduced g-ratio (Fig. 3B,C, right panels).These results corroborate that EPO overexpression influences the myelin formation rate, accelerating ensheathment around the axons.www.nature.com/scientificreports/

EPO stimulates early postnatal oligodendrocyte precursor cells maturation and activates the Erk1/2 pathway
After reporting EPO's transient function in developmental myelination, we proceeded to evaluate the role of EPO in the postnatal maturation of the oligodendroglial lineage in the SVZ.We previously evaluated the postnatal expression of EPORs (Fig. 4), followed by an analysis of OL maturation in EPO-overexpressing mice (Fig. 5), and in mice with EPORs target deletion from the OL lineage (Sox10) (Sox10-cre;EpoR fx/fx mice) (Fig. 6).Analysis of EPORs expression was performed in our two studied mouse models with fluorescent in situ hybridization (RNA Scope) of Epor mRNA in the SVZ and in OL transcription factor 2 expressing cells (Olig2 +), which is expressed throughout the entire OL lineage 35 .Brains of WT and Tg21 mice were evaluated at the postnatal ages P3, P7, P11, and P21 (Fig. 4A,B).We observed in WT and Tg21 genotypes that Epor expression in the SVZ of the third ventricle peaks at postnatal days P7 and P11 (Fig. 4A,B, the 2-way ANOVA, F(3,24) = 19.7,P < 0.0001).Epor expression, at P7, is two-fold higher in Tg21 mice than in WT mice (Fig. 4A,B, the 2-way ANOVA, F(1,24) = 11.2,P = 0.0027).Furthermore, combining FISH and immunofluorescent labelling of the Olig2+, at P7, Epors expression in Tg21 mice increased in Olig2+ cells (Fig. 4C, unpaired t-test, P = 0.0070, df = 24, and Fig. 4D), indicating a period for EPO signaling in developing OLs.
Next, we assessed Epor expression in Sox10-cre;EpoR fx/fx mice animals at P7 (Fig. 4E-H).Cre+ animals showed reduced Epor expression in the SVZ (Fig. 4E), cerebellum, and striatum, but not in the cerebral cortex and hippocampus (Fig. 4G, the 2-way ANOVA, F(1,30) = 23.64,P < 0.0001).In Cre+ animals, a reduction of Epor expression was quantified in Olig2+ cells in the SVZ (Fig. 4F,H, unpaired t-test, P = 0.0017, df = 30).Thus, the conditional deletion of Epor in the OL lineage was confirmed by the reduction in expression levels and allowed us to assess the impact of EPO signaling on OL maturation.
EPO regulation of postnatal oligodendrogenesis was morphologically analyzed in both our genetic mice lines in Olig2+ cells.OL proliferation was analyzed in the WT and Tg21 mice in the SVZ by double immunostaining Olig2 and the proliferation marker Ki67 at P7 and P11 (Fig. 5A,B).A higher number of total proliferating OL cells was quantified at P7 (Fig. 5B, the 2-way ANOVA, F(1,16) = 7.079, P = 0.0171); thus, constitutive EPO overexpression causes increased OL proliferation early in postnatal development.OPC originating in the SVZ migrate into the corpus callosum (CC), striatum and fornix to differentiate into mature myelinating oligodendrocytes 36 .Because the third wave of OPC maturation begins postnatally at day 4 (P4) after generation at P0 37 , and peaks at P15-21 in the CC 38 , we evaluated OL maturation in the CC at postnatal ages P7, P11, and P14 (Fig. 5C-H).Quantification of Olig2+ cells showed a significant genotype effect at P7 (Fig. 5C,D, the 2-way ANOVA, F(2,24) = 16.43,P < 0.0001).OL maturation was analyzed by double staining of Olig2, together with the mature lineage-specific marker (CC1) 39 .An increase in CC1+ cells was observed in the Tg21 mice at P7 (Fig. 5E,F, the 2-way ANOVA, F(1,24) = 6.867,P = 0.015).The increase in mature OLs caused by EPO in the CC could have resulted from a positive cell intrinsic drive to maturation, or an increase in survival.Finally, we assessed the number of MBPexpressing cells in the striatum, an area in which axons run less dense and in parallel, allowing them to be easily distinguished for MBP analysis.We observed an increased number at P7 and P11, showing that EPO increases the number of mature myelinating OLs (Fig. 5G,H, the 2-way ANOVA, F(1,24) = 14.94,P = 0.0007).
Next, the effect of EPORs deletion from the OL lineage (Sox10) on postnatal OL maturation was analyzed (Fig. 6).To confirm that the Cre-mediated deletion of EPOR in Sox10+ cells was specific to OLs, a Sox10-tdTomato reporter mouse line was first used.In this mouse line, tdTom expression was restricted to OLs (Olig2+ cells) and abundantly present at P7 in the SVZ (Fig. 6A).Sox10-tdTom cells were also distributed in the striatum partially colocalized with Olig2+ cells (Supplementary Fig. S1B); and a scattered distribution was found in the striatum and cortex colocalized with vascular pericytes (CD13) 40 derived from the neural crest 41 (Supplementary Fig. S1C).Because Sox10-tdTom+ cells colocalize primarily with Olig2+ cells, our work was specifically aimed at assessing the maturation of Olig2 lineage OPCs.Triple immunofluorescence staining for Olig2, together with OPC marker glycoprotein NG2 42 and mature OL-specific marker (CC1) at P7 (Fig. 6B-F), showed a reduction in the total expression of Olig2+ cells in the Cre+ samples (Fig. 6C, Unpaired t-test, P = 0.0058, df = 10).While CC1+ mature OLs were also significantly reduced (Fig. 6E, Unpaired t-test, P = 0.0002, df = 10), a higher number of NG2+ cells were quantified in Cre+ mice (Fig. 6D, Unpaired t-test, P = 0.055), suggesting an effect of EPOR inactivation in OL maturation and consequently a proportionally higher number of immature OLs (Fig. 6F, the  , red) and the axonal marker heavy neurofilament (NF200, green) in the striatum of WT and Tg21 mice at postnatal ages P7, P11, and P14.A higher myelination ratio observed by co-localization (yellow), occurs in the Tg21 striatum at P7 and P11.Scale bar: 20 μm.(F) Myelination axonal coverage (MBP/NF200) in WT and Tg21 mice.P7, unpaired t-test, ***P = 0.0005.P11, unpaired t-test, *P = 0.041.P14, unpaired t-test, P = 0.179, N = 6 animals per age and genotype.◂ 2-way ANOVA, F(1,20) = 221.3,P < 0.0001).We further investigated myelination and total Olig2+ cell number in the striatum at postnatal age P11 by double immunofluorescence staining for Olig2 and MBP (Fig. 6G-I).At P11, a reduction in Olig2+ cell number (Fig. 6G,H, unpaired t-test, P = 0.013, df = 8) and MBP expression (Fig. 6G-I, Unpaired t-test, P = 0.0017, df = 8) was observed in Cre+ mice as compared to Cre− mice, suggesting a reduction in mature myelinating OLs.Given that EPO is an anti-apoptotic cytokine 12 and that we previously showed that it promotes survival in hippocampal neurons during postnatal development 28 , we analyzed apoptosis (cleaved caspase 3) of Olig2+ cells of Cre+ and Cre− mice early at postnatal age P4.We observed increased OL apoptosis  (cleaved caspase3+/Olig2+ cells) in Cre+ mice (Fig. 6J,K, unpaired t-test, P = 0.0044, df = 6).Thus, the lack of EPORs in immature OLs at an early stage of postnatal development increases apoptosis, causing a reduction in total Olig2+ cell numbers being a second possibility for the reduced number in CC1 cells.

EPO increases key pro-differentiating, pro-myelinating, and Nfatc2/calcineurin pathway transcripts in postnatal OPCs
We evaluated the expression of major pro-differentiating and pro-myelinating transcription factors in our two mice models.So far, most transcripts reported to be stimulated by EPO in OLs (mostly in injury) involve PDGFαR and MBP 16,46 .Here, we evaluated the expression of several transcription factors (see Table 3) identified as major determinants of embryonic and adult OL differentiation and myelination in our two animal models 47 .We also evaluated the expression of transcription factors from the Nfat/calcineurin signaling, an activity-dependent regulator of OPC differentiation and myelination in rodents and humans 48 .

EPO stimulates postnatal motoric development, coordination, and learning
We performed behavioral experiments to evaluate how EPO brain overexpression and EPOR deletion from OPCs and Schwann cells (also regulated by Sox10 and responsive to EPO in their maturation and myelination) influences postnatal motoric development.Postnatal physical development, reflexes, and motoric development were examined in Tg21 and Sox10-cre;EpoR fx/fx mice lines to detect possible growth or physical development alterations.In both mouse lines, there was no difference from controls in physical development.Skin pigmentation (P3-P4), fur appearance (P6-P7), and incisor eruption (P7-P9) occurred at similar ages (Fig. 8A,B).The two lines slightly differed in their postnatal development in the eye-opening period (days).In the Sox10cre;EpoR fx/fx mouse line (mixed background), eye-opening occurred between days P11-P14, and in the Tg21 mice www.nature.com/scientificreports/line (Bl6C57 background) between P12-P15.The onset of reflexes was not different between mutated animals and their controls (Fig. 8C,D).Next, we tested the transition from pivoting to crawling and walking.In Tg21 and WT mice, pivoting was already established at P4, crawling started at P5, and was fully acquired by P7 in WT and P6 in Tg21 mice.Quadrupedal locomotion was first detected at P8 in WT and Tg21 pups and was normal in all WT pups at P12 and all Tg21 pups at P11.Therefore, complete motor development occurred one day earlier in Tg21 than control (Fig. 8E, the 2-way ANOVA, F(2,56) = 3.188, P = 0.048).In the Sox10-cre;EpoR fx/fx mice, pivoting started at P3 and was already established at P5, and crawling started at P5 and was fully acquired by P8 in all pups.Quadrupedal locomotion was first detected at P7, was normal at P11 in all Cre− pups, but was normal in all Cre+ pups only at P13.Thus, some Cre+ mice animals showed two days delayed in completing their motor development (Fig. 8F).Together, our data suggest that EPO CNS overexpression stimulates the development of the motor system.
After animals were able to walk, locomotion speed was assessed in a motoric open field test, measuring the time (maximal 30 s given time) needed to exit a circle in the center of an arena, performed between P8 and P14.A larger fraction of Tg21 mice than WT mice was able to perform the test, and the average time needed to exit the circle was shorter in Tg21 mice, reaching statistical significance at P9, P10, and P11 (Fig. 8G, the 2-way ANOVA, F(1,169) = 30.98,P < 0.0001).In Sox10-cre;EpoR fx/fx mice, a larger fraction of Cre+ animals were unable to perform the open field test (Fig. 8H, the 2-way ANOVA, F(1,80) = 6.77,P = 0.011).Thus, our data further indicate that EPO brain overexpression stimulates motor coordination but EPOR deletion from the Sox10 line delays it.
The effects of EPO on motor coordination were further tested with a rotarod apparatus at P14, P21, and P60.The results showed a significant effect of genotype and age, with Tg21 mice having a longer latency to fall (better performance) at P14 and P21 than WT (Fig. 8I, the 2-way ANOVA, F(1,114) = 27.48,P < 0.0001).At P14, the difference was mainly apparent during the late trials, suggesting better learning performance by the mutant mice (Fig. 8J, the 2-way ANOVA; F(1,152) = 35.36,P < 0.0001).Likewise, motor learning was better in Tg21 mice at P21, reaching a ceiling effect in the third trial (Fig. 8K, the 2-way ANOVA; F(1,152) = 9.561, P = 0.0034).Therefore, EPO overexpression stimulates postnatal motor coordination and motor learning.The effects of EPOR deletion from the Sox10-cre line showed an impact at P14, with Cre+ pups unable to perform the test (Fig. 8L, the 2-way ANOVA, F(1,62) = 1.776,P = 0.1875).At P14, in Cre+ pups, the motor coordination learning process across trials was impaired (Fig. 8M, the 2-way ANOVA; F(1,112) = 21.46,P < 0.0001).At P21, motor learning was equal between Cre+ and Cre− pups (Fig. 8N, the 2-way ANOVA; F(1,60) = 0.02738, P = 0.8708).Thus, a transient motor coordination and learning impairment was observed in Sox10-cre;EpoR fx/fx mice.This might reflect the delay in the maturation of myelinating OLs and Schwann cells.Together, our behavioral data suggest that EPO/ EPOR signaling stimulates motoric development and motor coordination learning.

Discussion
This study provides morphological, ultrastructural, molecular, and behavioral evidence for the crucial role of EPO in the normal (physiological) stimulation of developmental myelination in the mouse brain and improvement in motoric coordination learning.We show that EPO increases the proliferation of Olig2+ cells, prevents apoptosis, and stimulates maturation towards fully mature myelinating OLs.We show that Epors are highly expressed during the first two postnatal weeks in the SVZ and that EPO signals on postnatal OPCs activates the Erk1/2 pathway and the transcription from the Nfatc2 pathway and essential oligodendrocyte pro-differentiation and pro-maturation factors.
We show that constitutive EPO overexpression in the brain leads to a higher density of axons, an earlier onset of the myelination process, visible at P7, and a faster rate of axonal ensheathment, as seen morphologically and ultrastructurally.Notably, axons were myelinated independent of their diameter, while the number and consequent thickness of myelin sheaths were higher in Tg21 mice at an earlier stage in development.Thus, EPO increases the speed but not the extent of developmental myelination.We previously showed that EPO www.nature.com/scientificreports/overexpression increases postnatal synaptic density and accelerates neuronal maturation 28,29 , meaning that the earlier synaptogenesis and circuit maturation stimulated by EPO is accompanied by a faster myelination process and brain growth that reaches normality at earlier adulthood.Indeed, improved GABAergic development can act as a local environment cue to control myelination and OL lineage cell number 49 .The impact of EPO overexpression speeding up the process of brain myelination without alterations in adulthood suggests the potential for EPO administration in treating brain developmental hypomyelination.Indeed, systemic administration of EPO for 3 weeks in young adult mice at sufficiently high concentrations to cross the blood-brain barrier has been shown to increase myelin proteins 18 .EPORs are expressed in the developing mouse brain mid-gestation, localized in the neural tube and the neuroepithelium containing proliferating neuro-precursors 50,51 .Here we report a high level of Epor expression in OPC (Olig2+/Sox10+) in the SVZ and striatum at postnatal days P7 and P11, which markedly decreases in expression towards adulthood.At P7 an increase in Olig2+ cell proliferation is observed in our Tg21 mice.EPO has also been shown to stimulate OPC proliferation and differentiation in the SVZ in response to various types of insult, including prenatal ischemia 16 and demyelinating diseases 52,53 .However, treatments of EPO in juvenile mice (P28), showed no impact on the proliferation of hippocampal parenchymal OPCs 18 .EPOR expression in the hippocampus is not as high as in the SVZ area during early postnatal ages.Moreover, parenchymal OPCs respond differently than SVZ OPCs to growth factors such as (PDGF) 54 .Consequently, our model allows early postnatal stimulation of OPC proliferation and differentiation in the SVZ.A transient significantly increased expression of mature OLs (CC1+) and total Olig2+ cells was observed at P7 and P11 in Tg21 mice, indicating that EPO accelerates the process of oligodendrocyte maturation.Inversely, in the Sox10-cre;EpoR fx/fx mice line, SVZ OPC postnatal maturation in the Cre+ mice was delayed, reflected by a reduced number of CC1+ cells at P7.This reduction in mature CC1 was accompanied by reduced Epor expression in Olig2+ cells and increased apoptosis in the SVZ, the striatum, and the cerebellum.Analysis of local proliferation of Olig2+ cells showed an increase in proliferative OLs in Tg21 mice at P7. Thus, we suggest that EPO is proliferative and anti-apoptotic, and plays a physiological role in the maturation of OPCs, which adds a new function for EPO in postnatal brain maturation.The transient effects of EPO in development are regulated by the high expression levels of Epors.EPO's stimulation on astrocytes and microglia was not addressed in this study, two cell types that may also contribute to myelination control.In addition, in the Sox10-cre;EpoR fx/fx mice line EPORs from sparse neuronal crest-derived pericytes were also deleted in striatum and cortex, thus there may also exist potential interactions between pericytes and OPCs in perivascular regions 55 .
We have previously shown that mice overexpressing EPO in the brain activate the JAK2 Erk1/2 and AKT pathways postnatally 29 .In this study, we additionally elucidated those signaling pathways in the Sox10-cre;EpoR fx/fx mice line and determined a reduction in phosphorylation of Erk1/2 in the Cre+ mice.Thus, EPO signaling in OPCs activates the Erk1/2-MAPK pathway, a signal that promotes differentiation in OLs and Schwann cells as well as developmental myelination [43][44][45] .Schwann cell development and peripheral myelination may also be affected in our loss of function mice model.Erk1/2 inhibition was shown to induce re-myelination in vitro and animal models of acute experimental autoimmune encephalomyelitis 56,57 , but not in chronic demyelination processes 57 .Erk1/2 signaling is also a central regulator of immune cell function 58 .Thus, the process of re-myelination is influenced by the immune response.Conversely, during developmental myelination, Erk1/2 activation is influenced by growth factors, such as EPO, that stimulate differentiation and maturation of OPCs.We measured the expression of several key pro-differentiating and pro-myelinating transcription factors to determine which are stimulated by EPO to induce OPC to OL maturation and myelination.We provide evidence www.nature.com/scientificreports/EPO signaling induces postnatal terminal oligodendrocyte differentiation.We also provide the first evidence that EPO enhances the expression of Nfatc2 and calcineurin transcripts in OPCs.Nfatc2 is reportedly a highly expressed family member under the direct control of Sox10 in rodent oligodendroglial cells and coincides with increased calcineurin activation 48 .Moreover, calcineurin is usually activated by an increase in intracellular calcium levels that occur at the onset of OPC differentiation 63 , mediated by voltage-gated calcium or calciumpermeable glutamate channels 64,65 .Our previous data, showing enhanced synaptogenesis, fits with the observed increase in the activity-dependent transcripts and underscores the clinical value of upregulating Nfatc2 with EPO to stimulate oligodendroglial differentiation and remyelination.The observed increase in transcription factors with EPO and decrease without EPORs which could also result from the increased or decreased number in mature Olig2 cells.Thus, whether the EPO-stimulated Erk1/2 pathway controls the regulation of those transcripts remains to be determined.Finally, we show that EPO overexpression in the CNS leads to improved postnatal motor coordination and learning and that deletion of EPORs from OPCs and Schwann cells causes motoric impairment at P14. Higher myelination rates in the central and peripheric nervous systems are associated with improved motoric coordination, whereas hypomyelination with poor motoric coordination, likely due to the functioning of the myelin sheath on the efficiency of synaptic signals traveling along axons and the maintenance of high conduction speeds.Therefore, EPO's impact in postnatal motor coordination and motor learning most likely results from EPO stimulating developmental myelination in OLs and Schwann cells.Indeed, EPORs are expressed in Schwann cells after injury 66 , but expression during normal development requires further investigation.
In summary, our data fills an important gap in our knowledge about the physiological role of EPO in the developmental myelination process in the mouse brain.EPORs are developmentally regulated and stimulate differentiation of OPCs towards fully myelinating OLs.EPO accelerates developmental myelination and functionally improves motoric coordination and motoric learning without causing any alterations in total brain volume and myelination in adulthood.Considering that endogenous EPO production in the brain and EPORs expression are altered in preterm neonates [67][68][69][70][71] , our results explain, at least in part, the cause of impaired myelination in specific areas of the brain of preterm neonates.Similarly, our results strongly suggest that EPO is a promising pro-myelinating agent to overcome this impairment, although the efficiency may vary according to age and injury type and severity.
Tg21 mice were bred on a C57BL/6 background at the Laboratory Animal Service Center of the University of Zurich, and Sox10 and EPOR flox mice had a mixed background.
Animal experiments were approved by the Cantonal Veterinary Office of Zurich, Switzerland (license number 177/2016).Experiments were performed in accordance with the official guidelines and regulations, following the ARRIVE guidelines 73 .Animals were kept in standard housing conditions with food and water provided ad libitum.At least four male animals per genotype and age were collected from four different littermates and used for each experiment.The study concentrated on male animals due to their higher vulnerability to white matter injury.www.nature.com/scientificreports/

Tissue processing for H&E and Immunohistochemistry
Tissue from both transgenic lines and their controls was collected at postnatal days (P) P3, P7, P11, P14, P21, P25, and P60.For each specific experiment, different ages were taken, as specified in the results section.After anesthetization by i.p. overdose of pentobarbital (50 mg/kg i.p., Kantonsapotheke Zürich, CH, see Table 1 for volumes), mice were perfused with cold phosphate-buffered saline solution (PBS; pH 7.4), followed by paraformaldehyde (4%) (PFA) fixation.The brains were collected, sagittal cut along the brain midline, or kept intact for coronal cuts, and postfixed with 4% PFA at 4 °C for a time, varying according to age (see Table 1).Brains were then cryoprotected in 30% sucrose for 24 to 72 h at 4 °C until tissues sank and stored at − 20 °C.Brains were embedded in optimal cutting temperature (OCT) medium (VWR International) and cut in sagittal or coronal serial sections with a sliding microtome (MICROM HM 400, MICROM International GmbH, Walldorf, Germany).Thickness and series sample fraction (ssf) varied depending on mouse age (see Table 1).Tissues were stored at − 20 °C in an antifreeze solution until immunohistochemical staining.

H&E staining
A series of tissues at ages P3, P7, P14, and P25 were mounted on gelatin-coated glass slides (Thermo Scientific, Menzel, GmbH) and allowed to air-dry overnight at room temperature (RT).Tissues were immersed in dH 2 O for 1 min, followed by immersion in hematoxylin solution (MHS16, Mayer, Sigma-Aldrich Chemie GmbH, Germany) for 30 s and rinsed in water for 1 min.The step was repeated depending on the desired intensity.Tissues were then immersed in 1% eosin (E4009, Sigma-Aldrich Chemie GmbH, Germany) solution for 20 s.Slides were dehydrated in ethanol of increasing concentrations (2 × 70%, 2 × 96%, 3 × 100%) for 5 min each, followed by clearing in xylene 4 times for 5 min.On the following day, the sections were coverslipped with Eukitt mounting medium (Fluka analytical, Sigma-Aldrich Chemie GmbH, Germany), let to dry overnight, and longtermed stored at RT.

Immunoperoxidase staining
Myelinated areas in the brain were quantified in serial coronal sections at P3, P7, P11, P14, and P21 immunolabeled for myelin basic protein (MBP).Tissues were transferred from the antifreeze solution to a 12-well plastic plate filled with Tris-Triton (pH 7.4) for washing; tissues were then incubated with primary antibody overnight at 4 °C under constant agitation in a solution containing 0.2% Triton X-100 and 2% normal serum (NS).Following three washes, a biotinylated-conjugated antibody (goat anti-rabbit, Jackson Immunoresearch, 1:300) was applied and incubated for 30 min, at RT, in a moist chamber under continuous agitation.Brain tissues were then washed in Tris-Triton three times and incubated in Avidin-Biotin Complex (ABC) solution (Vectastain Elite ABC Kit standard, Vector Laboratories, Burlingame USA) under continuous agitation for 30 min at RT and rinsed again three times.Tissue sections were pre-incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB)-containing Tris-Triton with 0.05% Triton X-100 for 5 min under agitation; the reaction was then started by adding DAB solution containing 0.01% hydrogen peroxidase.After 5-7 min, depending on the intensity of the staining, the reaction was stopped by transferring the sections into ice-cold PBS, followed immediately by another three washing steps.The sections were mounted on gelatin-coated glass slides and left to dry overnight.On the following day, they were dehydrated, coverslipped with Eukitt mounting medium, and long-termed stored at RT.

Immunofluorescence stains
Double or triple immunofluorescence staining at P7, P11, and/or P14 was used to analyze multiple markers within the same section.The tissues were double stained for analysis of myelinated axons (NF200 and MBP), OL proliferation (Ki67 and Olig2), myelinating OLs (MBP and Olig2), OL apoptosis (Caspase 3 and Olig2), and Sox10-tdTom cell expression specificity (tdTom and DAPI).The tissues were double (CC1 and Olig2) or triple stained for analysis of OL maturation (NG2, CC1, and Olig2).Free-floating sections were washed 3 times before incubating with the first antibodies (see Table 2) overnight at 4 °C under continuous agitation in a solution containing 2% Triton X-100 and 2% NGS.After washing the sections three times, they were incubated with the secondary antibodies raised in donkey against the different species of the primary antibodies and coupled to either Alexa488, Cy3, or Alexa647 in a solution containing DAPI and 2% NGS, at RT for 30 min in the dark.
After another washing step of 3 times, sections were mounted on gelatin-coated glass slides, coverslipped with Dako fluorescence mounting medium (Dako, Carpinteria CA, USA), and stored in a closed cardboard box at 4 °C.FISH of murine Epor (RNAscope Probe-Mm-Epor) was performed in Sox10-Cre Tg/+ /EpoR fx/fx (cre+), and Sox10-Cre +/+ /EpoR fx/fx (cre-) animals at postnatal ages P7 and P11, using the RNAscope Multiplex Fluorescent Reagent Kit v2 from Advanced Cell Diagnostics and the Opal 520 fluorophore from Perkin Elmer as previously described in our protocol 28 .To quantify Epors on OLs, FISH was done followed by immunolabeling of Olig2 cells (see Table 2).

Electron microscopy (EM)
Ultrathin tissue sections were prepared from P11 and P14 male mice for transmission electron microscopy (TEM) imaging, according to our previously described protocol 29 .Six animals per age and genotype were used for the analysis.

Image acquisition for H&E, immunostaining, FISH, and EM analysis
Sections processed for H&E and MBP immunoperoxidase staining were analyzed with the MCID™ software, using calibrated images for normalization.Images were visualized and digitized using a precision illuminator (Northern Light Model B95, Imaging Research Inc., Brock University, St. Catharines, Canada) and Cool SNAP cf photo-camera (Photometrics, Tuscon, AZ, USA) with a Micro-Nikkor (55 mm + 12 mm) objective (Nikon Corp.).All double and triple immunofluorescent and FISH stainings were imaged with a Zeiss LSM 700 or 900 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) with a 25X or a 40X oil immersion objective with a numerical aperture (NA) of 1.4, a scan zoom of 1X and an image size of 1024X1024 pixels.Immunofluorescence images from stratium and corpus callosum areas were taken as z-stacks (10-15 sections at 1 μm intervals).
FISH images from SVZ were taken as z-stacks (6 sections with 0.5 μm intervals).All images were recorded with sequential scanning to avoid overlapping of the emission spectra, and the confocal pinhole was set to 1 airy unit.Four different sections were taken per animal and condition.For statistical analysis, all sections were averaged per mouse.
The region of analysis for axonal myelination was the striatum.The areas of interest for OL differentiation were the subventricular zone of the third ventricle, the corpus callosum (CC), and the striatum.FISH samples were imaged in the subventricular zone and colocalized with Olig2 staining.
For EM, semithin sections of posterior white matter were observed with a bright field microscope.Then EM micrographs of the caudal white matter were taken with the 100 kV transmission electron microscope (TEM-Philips CM100 and Telos) connected to a digital CCD camera.

Brain volume analysis
Data analysis was done using the MCID software (MCID Elite 6.0, InterFocus Imaging Ltd., Cambridge, UK).Four pups from different breeding pairs were analyzed per genotype and age in sagittal serial sections throughout one hemisphere.Serial sampling fraction (ssf) varies according to age, as specified in Table 1.
The total volumes (V tot ) of the prosencephalon, dorsal pallium (cortex), middle pallium (hippocampus), and diencephalon (TH and HTH) were calculated from the areas delineated (Fig. 1D, 74 ) in every section as follows: where: A i -A n ; n = number of sections analyzed, and the section thickness (h)

MBP optical density analysis
Using the MCID software (MCID Elite 6.0, InterFocus Imaging Ltd., Cambridge, UK), MBP immunoperoxidase staining intensity was assessed by densitometry analysis.Gray value calibration (Kodak step tablet no.310ST607) was performed, and the intensity was measured in the different regions of interest: cortex, striatum, and CC.Each sample was corrected for background staining variations by subtracting the intensity value measured in an unmyelinated area.

Immunofluorescence image analysis
The degree of axonal myelination was determined in double-immunofluorescence images for MBP and NF200 with the Software Imarisx64, 8.3.0,Bitplane AG, CH. (Center for Microscopy and Image Analysis, Zurich), by identifying the surfaces of NF200 and MBP through the "surface" function and setting the threshold and number of voxels to discriminate between signal and background noise.Subsequently, 3D figure reconstruction was done with the "wizard" function with surface and ratio automatically generated by the program.Once 3D reconstructions were done for both channels, the "surface co-localization" tool linked to Imaris Xtension was run, giving the surface-to-surface co-localization area covered by MBP over Nf200 normalized to the field of view.
For image analysis of OL proliferation, OL apoptosis, OL maturation, myelination (MBP), and Sox10-tdTom expression, maximum intensity projections from consecutive images of a z-stack were created using Fiji (ImageJ imaging software, National Institutes of Health, Bethesda, USA).Stack images from Olig2 and MBP single stains were converted to 8-bit greyscale before proceeding, and the threshold was manually subtracted to discriminate between signal and background noise.Then, total Olig2+ numbers per field of view were automatically counted with the Fiji Analyze Particles tool.Merged cells were cut by adding a 1-pixel thick line.Size particles were adjusted between 5-Infinity mm 2 and circularity from 0.1 to 1.0.In the display, results were controlled so that each outline choice corresponded to one individual cell.MBP cells were quantified manually with the cell counter plugin.Double Ki67/Olig2 stained cells were identified with the Fiji colocalization threshold plugin.Before colocalization, color channels were made composite, and the background was subtracted manually for each channel.A new greyscale image, including the colocalized pixels above their respective thresholds, was created, and the colocalized cells were counted with the Fiji Analyze Particles tool as described for Olig2.Double NG2/ Olig2 and CC1/Olig2 stains were quantified manually with the cell counter plugin.At least three serial sections of the SVZ and corpus callosum were quantified.

FISH analysis
Analysis was done in z-stack images with maximum intensity projection using a custom-made cluster analysis macro in Fiji/ImageJ (NIH) software.Processing of Epor particles (green channel) was separately analyzed, with background subtraction using rolling ball radius, Gaussian blurring, and thresholding for selecting regions of interest of high staining intensity, as well as shape (0.5-1 circularity) and size (0.1-1 μm diameter) restrictions.The same parameters were used in all images per genotype and age.Four animals and three images per animal were used for the analysis across brain areas.Colocalization of Epors with Olig2+ stained cells was identified with Fiji.Once Epor particles (green channel) were processed as previously described, a new merged image with both channels was made.Olig2+ cells (magenta channel) were identified based on the nucleus centroid and an area of a nucleus-cytoplasm ratio of 0.6 was selected for quantification of the Epor particles (Supplementary Fig. 3).Epor dots were made binary and counted with the Analyze Particles tool with shape (0.5-1 circularity) and size (0.1-1 μm diameter) restrictions.

Electron microscopy (EM) analysis
Axon diameter and myelin sheet thickness were determined in images from electron microscopy captured with 17,500× magnification.Axon diameter was the average of the largest and smallest diameter, and myelin sheet thickness was determined by the number of turns around the axon and the g-ratio (g) = di/do, where: di = axon diameter without myelin; do = total axon diameter.

RNA preparation and quantitative real-time PCR (RT-qPCR)
Tissue containing the striatum and the subventricular zone was collected from our two transgenic mice lines and controls at P4 and P11: N = 8 male animals per age in Tg21 and N = 5 per age in Sox10-cre;EpoR fx/fx mice line.Total RNA was extracted using the ReliaPrep RNA Tissue Miniprep System (Promega, #Z6110) according to the manual.DNase I digestion was used to eliminate DNA contamination.RNA purity and quantity were determined spectrophotometrically (Nanodrop 2000, ThermoScientific).First-strand cDNA was obtained by reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, #K1622).Samples (10 ng/µl cDNA) were analyzed by SYBR Green (ThermoFisher Scientific, #A25741) semi-quantitative real-time PCR (qRT-PCR) (7500 Fast Real-Time PCR System, ThermoFisher Scientific).Primers for mRNA expression analyses were designed with Primer 3.0.Software to amplify human or murine genes without crossspecificity (see Table 3).Oligo properties were calculated using Oligo Analyzer 3.1.mRNA expression levels were calculated using the ∆∆Ct method 75 and normalized to beta-actin (βACT).Each group of samples was normalized to WT P4.

Developmental milestone tests
We evaluated the postnatal development of mice following a battery of tests examining weight gain, physical development, the appearance of reflexes, and motoric development throughout the first 21 days of life 76,77 .
Physical Appearance includes skin pigmentation, fur appearance, incisor eruption eye-opening.Reflex Appearance consists of the righting reflex and cliff avoidance in which the vestibular and locomotor systems and the body strength and coordination of the animals are evaluated; negative geotaxis, which evaluates the vestibular/ labyrinthine system of the animals; and vestibular drop, in which the vestibular system and body strength are tested when the animal is suspended by its tail and raises its head to the level of the hind limbs by arching its body sideways.All these reflexes were tested for 30 s in N = 10 mice per genotype.Motoric development was evaluated by monitoring the age at which the mice started pivoting, crawling, and walking.The open field test evaluates the speed of quadrupedal locomotion in a circle with a diameter of 13 cm, and the time needed to leave the circle was registered.The maximum given time for the test was 30 s. Tests were done on 6 to 15 animals per genotype.

Motor coordination and motor learning
Using the rotarod performance test, based on a rotating rod suspended above a cage floor, high enough to induce avoidance to fall but not injury, we tested motoric coordination at P14, P21, and P60, and motoric learning at P21. Motoric coordination was tested in P14 mice four times for 4 min with 5 min recovery intervals.The initial Table 3. Primers used for RT-qPCR.rotation speed was set to 1 revolution per min (rpm), with an acceleration of 10 rpm within 4 min.Motoric coordination in P21 and P60 mice was tested four times for 5 min with 5 min recovery intervals.The initial rotation speed was 4 rpm and acceleration to 45 rpm within 5 min.An activity control test was performed four times with a constant speed of 10 rpm for 5 min.The time to fall and speed were recorded in each test.Tests were done on 8 to 20 animals per genotype and age.Tests were done on 10 to 20 animals per genotype and age.

Experimental design and statistical analyses
Data collection was conducted by laboratory members who were blinded to the genotypes.Tissues from mice were collected at postnatal ages (P): 3, 7, 11, 14, 21, and 25 for morphological evaluation of brain growth, myelination, and OL precursor cell (OPCs) differentiation and maturation.In addition, tissue was collected at P9, P11, and P14 for ultrastructural evaluation of myelin ensheathment with electron microscopy.Fresh tissue was also collected at P7 for Epors expression analysis, at P4 and P11 for pro-differentiation and pro-myelination transcript analysis, and WB analysis in Sox-cre mice.No more than one male pup per age was taken from each breeding group.Six different breeding groups were used per genotype, and their controls for behavioral analysis, and tissue was collected afterward.Statistical analyses and graphs were performed using GraphPad Prism 9.0.0 (GraphPad Software, San Diego, CA, USA).We used scatterplots with bars to present single data points and the mean ± standard deviation (SD).Parametric data with one condition (genotype) were analyzed using an unpaired, two-tailed Student's t-test.A two-way ANOVA with multiple comparisons post hoc test was used to compare data influenced by two factors (e.g., age and genotype or brain areas and genotype).Normality was tested with Kolmogorov-Smirnov (KS) test.The difference in relative frequency distributions between WT and Tg21 was tested with a 2-sample Kolmogorov-Smirnov (2 s-KS) test using SPSS 28.0.1 (IBM).A P value < 0.05 was considered statistically significant for all statistical tests.To confirm appropriate sample sizes for experiments, a power analysis was conducted using G*Power software (Heinrich Heine Universität, Düsseldorf, Germany) 78 .The F ratio and the exact P value for multiple comparisons are described in the figure legends.

Table 1 .
Perfusion and brain cutting thickness parameters.

Table 2 .
Primary antibodies were used.